In a spectrophotometer a beam of light of a selected wavelength or frequency is passed through a sample where some of the light is absorbed by the molecules comprising the sample. The light which passes through the sample is received by a light sensitive detector system such as a photometer. The less light energy that is absorbed by the sample results in more light being received by the light detector system. The detector system generates an electrical signal of a strength proportional to the intensity of the light it receives. The output of the light detector system, for example one utilizing a photomultiplier tube, is generally an analog current signal proportional to the light intensity received, which thus is proportional to the light transmittance of the sample.
The light detector system generally has an amplifier, such as an operational amplifier, to convert the analog current signal from the light detector to an analog d.c. voltage signal. The d.c. voltage signal is processed by additional electronics and applied to a display, such as a chart recorder, to provide a visual and/or permanent record of the sample light transmittance, i.e., absorbance (absorbance=log transmittance) at a selected light wavelength or through a wavelength scan.
The selected wavelength light beam is generated from a generally white light source through the use of a monochromator. The monochromator provides a monochromatic beam of light having a range of wavelength within a narrow controlled spectral band. This is generally accomplished by dispersing the white light received into a sweeping spectrum of differing wavelengths of light by directing the white light through a prism or reflecting the light from a dispersion grating. The monochromatic light wavelength generated by the monochromator is selected by rotating the prism or grating to direct light of the desired wavelength in the spectrum through a narrow slit or aperture out of the monochromator. The remaining light of undesired wavelengths is not permitted to pass from the monochromator. Thus, by rotating the dispersing element the light spectrum can be moved across the narrow slit to obtain a selected wavelength monochromatic light for application to the sample.
The spectral bandwidth of the monochromatic light generated by the monochromator is determined by the width of the slit, the dispersion function of the dispersing element, and the rotational location of the dispersing element relative to the slit. Change in wavelength of the monochromatic light generated, however, usually does not have a linear relationship with change in the angular position of the dispersing element. That is, the wavelength light generated is not a linear function of the dispersing element's change in angle of rotation. Generally, the wavelength of light generated by the monochromator is related to the rotational position of the dispersing element by the following formula: EQU .lambda.=K sin .theta. (1)
where,
.lambda.=wavelength of monochromatic light PA1 K=dispersion constant, e.g. grating constant PA1 .theta.=angular position of dispersing element (from a base position
In order to obtain accurate performance of the spectrophotometer in analysis of a sample it is very important to be able to accurately generate a select and stable monochromatic light beam for application to the sample. This requires repeatable and precise positioning of the dispersing element in the proper angular position. Manufacture of the instrument components including the dispersing element and its positioning mechanism, and assembly of these components in the instruments, must be performed with very high precision. A complex, highly precise, and expensive mechanism is necessary to direct the dispersing element. Furthermore, the nonlinear relationship between the rotational change of the dispersing element and the change in wavelength of the monochromatic light generated additionally complicates the positioning mechanism design. Prior designs have incorporated complex linkages, precision cams, and specially designed gears to accomplish approximation of linearity between rotation of the dispersing element and wavelength selection in order to obtain accuracy repeatability and ease of use.
With these designs costly and time consuming calibration procedures are necessary to assure proper optical alignment of the assembly and to correct manufacturing variances in the optical elements. Failure to provide either intensive quality control or calibration would often result in the spectrophotometer which was unable to accurately analyze, or reproduce accurately the analysis of, the substances for which it was designed.
A method and apparatus for performing a rapid, accurate and automatic calibration of a scientific instrument utilizing select monochromatic light generated by a monochromator is described herein. The described method and apparatus resolve the problems associated with calibration of scientific instruments discussed above to assure that the instrument consistently performs its function with high accuracy. This calibration procedure utilizes an absorption medium which is placed at or near a sample position to provide information relating the function of the monochromator to the light wavelength generated. Though accurate and reliable in performing instrument calibration, i.e. monochromator calibration, the calibration procedure described cannot overcome errors due to improper care in cleaning of the sample chamber of such an instrument. Since the absorption medium is placed within the sample compartment, any sample residue remaining in the sample compartment which absorbs light could affect calibration procedure. This is particularly critical with the described procedure since the computer in performing calibration is searching for an absorbance event to determine the assignment of a light wavelength to a dispersing element angular position. A false indication of absorbance would result in an incorrect angular position being assigned to a certain wavelength, and invalidate instrument calibration.
A need thus exists in the field of scientific instrumentation which utilizes varying wavelength monochromatic light for a select purpose, and in the manufacture of spectrophotometers in particular, to resolve the difficulties associated with their calibration when interfering substances are permitted to remain in the sample chamber and to provide accuracy and reproducibility in their function through effective calibration.